Exploring rose absolute and phenylethyl alcohol as novel quorum sensing inhibitors in Pseudomonas aeruginosa and Chromobacterium violaceum

Inter-cellular signaling, referred to as quorum sensing (QS), regulates the production of virulence factors in numerous gram-negative bacteria, such as the human pathogens Pseudomonas aeruginosa and Chromobacterium violaceum. QS inhibition may provide an opportunity for the treatment of bacterial infections. This represents the initial study to examine the antibiofilm and antivirulence capabilities of rose absolute and its primary component, phenylethyl alcohol. QS inhibition was assessed by examining extracellular exopolysaccharide synthesis, biofilm development, and swarming motility in P. aeruginosa PAO1, along with violacein production in C. violaceum ATCC 12472. Molecular docking analysis was conducted to explore the mechanism by which PEA inhibits QS. Our results indicate that rose absolute and PEA caused decrease in EPS production (60.5–33.5%), swarming motility (94.7–64.5%), and biofilm formation (98.53–55.5%) in the human pathogen P. aeruginosa PAO1. Violacein production decreased by 98.1% and 62.5% with an absolute (0.5 v/v %) and PEA (2 mM). Moreover, the molecular docking analysis revealed a promising competitive interaction between PEA and AHLs. Consequently, this study offers valuable insights into the potential of rose absolute and PEA as inhibitors of QS in P. aeruginosa and C. violaceum.


GC-MS analysis
The principal compounds in rose absolute were identified using gas chromatography-mass spectrometry (GC-MS).An Agilent 7890B/5977B GC/MS system with a ZB-WAX plus column (60 m × 250 µm, 250 nm; Phenomenex) was used.Helium (≥ 99.999%) was the carrier gas at a constant flow rate of 1.2 mL/min.Samples (1.0 µL) were injected in split mode (2:1, 250 °C).The ion source temperature was set to 150 °C with electron impact ionization at 70 eV.

Antibacterial assay
The antibacterial properties of rose absolute (dilutions: 1:5, 1:10, 1:20, 1:100 v/v) and PEA (16, 8, 4, 2 mM) were assessed using well-diffusion assays.P. aeruginosa and C. violaceum were cultured in LB broth at 37 °C for 24 h.Cultures were adjusted to the McFarland no.0.5 standard.LB agar (0.3% w/v) was inoculated with 100 µL of bacterial culture and poured onto pre-warmed LB agar plates.Wells were created using a sterile cork borer, and 50 µL of rose absolute or PEA was added.Plates were incubated at 37 °C for 24-48 h, and zones of inhibition (mm) were measured.DMSO served as the negative control, and gentamicin (10 µg/ml) was the positive control.

MIC assay
The minimum inhibitory concentrations (MICs) of rose absolute and PEA were determined.Twofold serial dilutions (rose absolute: 4% to 0.03% v/v; PEA: 16 to 0.125 mM) were prepared in LB broth 41 .Tubes were inoculated and incubated at 37 °C for 24-48 h.The MIC was defined as the lowest concentration with no visible bacterial growth.Experiments were replicated three times.

The EPS production
EPS production was measured using the Sulfuric Acid-UV method 44 .Bacterial cultures were treated with 3 mL of sulfuric acid, vortexed, chilled on ice, and the optical density was measured at 315 nm.

Antibacterial effects
In this study, we examined the antibacterial properties of rose absolute and PEA against Pseudomonas aeruginosa (PAO1) and Chromobacterium violaceum ATCC 12472.
Previous studies have documented the antibacterial efficacy of rose absolute and PEA against various Gramnegative and Gram-positive bacteria 30,39,49 .In this study, we examined the antibacterial characteristics of rose absolute and PEA against P. aeruginosa and C. violaceum.Rose absolute exhibited significant antibacterial potential against C. violaceum (Table 2 and Fig. 1).The maximum inhibition zone for rose absolute was 20 ± 0.7 mm, with MIC values of 1.0% (v/v) against C. violaceum.PEA and DMSO (control) did not exhibit inhibitory effects on either bacterial strain.

Inhibition of P. aeruginosa swarming motility by rose absolute and PEA
Bacterial motility plays a crucial role in biofilm formation, and the swarming ability of P. aeruginosa depends on type IV pili and flagella 53 .Compromised swarming ability correlates with reduced biofilm formation 54 .Citronellol, geraniol, and nerol have been reported to inhibit swarming motility in P. aeruginosa 55 .We found that rose absolute (0.5% v/v) and PEA (2 mM) inhibited swarming motility by 94.7% and 62.4%, respectively (Fig. 3).This study is the first to demonstrate that rose absolute and PEA inhibit swarming motility in P. aeruginosa PAO1.

EPS production
Extracellular polymeric substance (EPS) is crucial for biofilm structure, microcolony formation, and resistance to antimicrobial agents [56][57][58] .We demonstrated that sub-inhibitory concentrations of rose absolute and PEA reduced EPS production in P. aeruginosa after 24 h of treatment.Using the sulfuric acid method, rose absolute and PEA significantly decreased EPS production by 60.5% and 33.5%, respectively (p > 0.05) (Fig. 4).Similarly, Musthafa et al. ( 2012) reported a decrease in EPS production by approximately 54% in P. aeruginosa PAO1 following treatment with phenylacetic acid 59 .

Violacein inhibition assay
Chromobacterium violaceum synthesizes violacein through a QS system controlled by the CviR-mediated QS mechanism 60 .An ideal QS inhibitor should not disrupt normal bacterial growth to prevent bacterial resistance 61,62 .We evaluated the anti-QS activity of rose absolute and PEA on violacein production in C. violaceum ATCC 12472.At sub-MIC concentrations, neither rose absolute nor PEA inhibited bacterial growth.Rose absolute and PEA inhibited violacein production by 98.1% to 94.2% and 62.5% to 6.7%, respectively, in a concentration-dependent manner (Fig. 5).Previous studies have shown that Capparis spinosa extract (2 mg/mL) inhibited violacein production by up to 88% 63 , that Psidium guajava and curcumin inhibited violacein production by C. violaceum 64,65 .Our results indicate that rose absolute (0.5% v/v) and PEA (2 mM) significantly decreased violacein levels by 98.1% and 62.5%, respectively (Fig. 5).

Molecular docking
Molecular docking analysis, a simulation technique, elucidates interactions between receptors (proteins, nucleic acids) and ligands (compounds) 66 .This computational approach generates scores that reflect potential energy changes upon the interaction between a protein and a ligand.Steric interactions, metal ions, and hydrogen bonds collectively influence the resulting score.As shown in Table 3, lower scores (more negative) signify stronger binding affinities 67 .In this investigation, molecular docking analysis was conducted to explore the anti-QS mechanism of PEA.We selected five distinct forms of the LasR protein (PDB ID: 2UVO, 6D6L, 6D6A, 6D6O, and 6D6P) and two variants of the RhlR protein (PDB ID: 4EY15, 4EY17) for analysis [67][68][69] .This analysis aimed to reveal potential mechanisms of QS inhibition by disrupting LasR and RhlR proteins.Detailed information regarding binding affinities and involved amino acid residues is presented in Table 3, supplemented by Figs. 6 and 7.
The structural differences between the LasR and RhlR proteins explain the various binding scores of PEA.Figures 6 and 7 illustrate that PEA interacts well with the LasR-6D6L and RhlR-4Y17 proteins, embedding fully in their active binding pockets.Among all the LasR proteins, PEA exhibited the highest binding affinity to the LasR-6D6L protein (Table 3).To elucidate the interaction between PEA and LasR proteins, we examined its interaction with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the natural ligand of LasR, and the interaction of furanone, a standard inhibitor, with LasR proteins.The binding affinities of PEA, OdDHL, and furanone to the LasR-6D6L protein were calculated as − 7.2, − 7.8, and − 6.6 kcal/mol, respectively.This finding suggests that PEA interacts with the LasR-6D6L protein with higher affinity than furanone.Although PEA's binding affinity to LasR-6D6L was lower than that of OdDHL, it acted competitively.
The RhlR-4Y15 and RhlR-4Y17 proteins showed similar outcomes (Table 3).Specifically, RhlR-4Y17 demonstrated the highest binding affinity with PEA, significantly surpassing that of furanone-RhlR-4Y17 and equaling the binding affinity of Bhl-RhlR-4Y17.Hydrogen bond interactions are pivotal in stabilizing the ligand significantly.Furthermore, pi-alkyl, van der Waals, and pi-pi interactions also hold substantial importance in ligand binding, enhancing the role of hydrogen bonds.Throughout this investigation, carbon-hydrogen bonds, conventional hydrogen bonds, van der Waals, pi-pi and pi-alkyl interactions emerged as predominant factors.
The findings demonstrate binding competition among PEA, OdDHL, and furanone for identical amino acid residues in all five LasR proteins.Moreover, PEA may bind to LasR proteins better than furanone.This observation also applies to RhlR's 4Y15 and 4Y17 proteins.Notably, the binding affinity of RhlR-4Y17 to its cognate ligand (N-Butanoyl-L-homoserine lactone (BHL)) is equal to its binding affinity to PEA, which is a significant www.nature.com/scientificreports/finding.Similarly, the binding affinity of CviR'-3QP1 to its cognate ligand (N-acyl-L-homoserine lactone (AHL)) is lower than its binding affinity to PEA, which is an impressive result.
The PEA ligand interacted with the CviR-3QP5 protein via the amino acid residues SER155, ILE153, THR140, MET135, ALA130, PHE126, PHE115, TRP111, LEU100, ILE99, ASP97, TYR88, TRP84, TYR80, LEU57.This region, where the 3QP5 protein has shown binding affinity, is consistent with data from Bodede et al. 70 .The binding affinity of the PEA ligand to the CviR-3QP5 protein was calculated as − 6.3 kcal/mol, which is equivalent to the binding affinity of the CviR-3QP5 protein to its natural ligand, AHL.Therefore, it can be said that PEA is a good inhibitor for CviR-3QP5.Similar results can be observed for the interaction of PEA with the CviR'-3QP1 protein.The amino acid residues to which the CviR'-3QP1 protein has binding affinity are detailed in Table 3.This region is similar to the active region of the CviR-3QP5 protein and aligns with existing research findings 70,71 .
Hydrogen bond interactions significantly stabilize the ligand.In addition to these, van der Waals, H-bonds, pi-pi, and pi-alkyl interactions are also significant in ligand binding.In this study, conventional hydrogen bonds, carbon-hydrogen bonds, van der Waals, pi-alkyl, and pi-pi interactions seem to dominate.The findings demonstrate binding competition among PEA and OdDHL for identical amino acid residues in all five LasR proteins.In addition, it suggests that PEA may bind to LasR proteins better than furanone.The same is true for RhlR's 4Y15 and 4Y17 proteins.The fact that RhlR-4Y17's binding affinity to the cognate ligand (BHL) is equal to its binding affinity to PEA is a noteworthy finding.Similarly, the binding affinity of CviR'-3QP1 to its natural ligand (AHL) is lower than its binding affinity to PEA.This suggests that the compounds compete for the binding site alongside the signaling molecule.

Conclusions
This study is the first to investigate the anti-biofilm potential of rose absolute and PEA against P. aeruginosa PAO1.Our findings demonstrate that rose absolute and PEA significantly inhibit QS-controlled processes, including EPS production, biofilm formation, swarming motility, and violacein production, without significantly affecting bacterial growth.Molecular docking analyses revealed that PEA interacts with LasR, RhlR, CviR, and CviR' proteins similarly to cognate AHLs, often outperforming the QSI furanone C30.These insights could guide the development of novel antivirulence therapeutic strategies against biofilm-associated infections.

Table 1 .
Chemical composition of rose absolute analyzed by GC-MS.

Table 3 .
The findings from the molecular binding analysis of various LasR and RhlR structures from P. aeruginosa PAO1, along with CviR and CviR' proteins from C. violaceum ATCC 12472, with PEA, furanone C30, OdDHL, BHL, and AHL are presented.